Breaking the bottleneck: Eukaryotic membrane protein expression for high-resolution structural studies

Midgett, Charles R.; Madden, Dean R.

doi:10.1016/j.jsb.2007.07.001

Cited by 83 publications

(62 citation statements)

References 118 publications

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“…[7][8][9] Because of concern about protein misfolding, glycosylation, membrane lipid composition, and aggregation when proteins are expressed in the higher yielding bacterial, yeast, or insect cells, eukaryotic expression in mammalian cells is often preferred. [10][11][12][13] Heterologous expression of Cys-loop channels in mammalian cell lines has been achieved but rarely in the quantities and purity required for structural studies. For example, the best success has come from the 5-HT 3A R, where efforts over decades 14,15 have lead to yields of 100 pmol/plate with a specific activity of 12-60 pmol/mg.…”

Section: Introductionmentioning

confidence: 99%

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors

et al. 2010

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The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABA A receptors containing a1 and b3 subunits could be expressed with specific activities of 29-34 pmol/mg corresponding to 140-170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT 3A ) receptors were 49-63 pmol/mg and 245-315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3-3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one Abbreviations: 5-HT 3 AR, 5-hydroxytryptamine-3A receptor; CMC, critical micelle concentration; DDM, n-dodecyl-b-D-maltopyranoside; EPR, electron paramagnetic resonance; GABA A a1b3R, gamma-aminobutyric acid type A receptor with a1 and b3 subunits; GPCR, G protein-coupled receptor; GlyR, glycine receptor; HEK293, human embryonic kidney cells; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; NMR, nuclear magnetic resonance; PEG, poly(ethylene glycol).Additional Supporting Information may be found in the online version of this article. Published by Wiley-Blackwell. V C 2010 The Protein Society step after solubilization using ANTI-FLAG affinity chromatography with yields of 30-40%. Purified receptors were functional. Binding of the agonist [ 3 H]muscimol to the purified GABA A R was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles.

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Section: Introductionmentioning

confidence: 99%

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors

et al. 2010

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“…The great benefit of choosing a eukaryotic host for overexpression of a protein of interest are the availability of a posttranslational modification system as well as the frequently enhanced protein folding (Midgett and Madden 2007). Eukaryotic proteins tend to misfold or lack biological activity when expressed in prokaryotic expression systems such as E. coli (Cregg, Cereghino et al 2000;Midgett and Madden 2007).…”

Section: Eukaryotic Expression Hostsmentioning

confidence: 99%

“…Eukaryotic proteins tend to misfold or lack biological activity when expressed in prokaryotic expression systems such as E. coli (Cregg, Cereghino et al 2000;Midgett and Madden 2007). To overexpress these proteins, different yeast strains, insect cells or even mammalian cell lines have been developed as expression hosts (Figure 1 -(I) expression, right side).…”

Section: Eukaryotic Expression Hostsmentioning

confidence: 99%

Rational and Irrational Approaches to Convince a Protein to Crystallize

Abts¹,

Schwarz²,

Tschapek³

et al. 2012

Modern Aspects of Bulk Crystal and Thin Film Preparation

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“…Membrane proteins account for 20-25 % of all sequenced open reading frames and play key roles in many essential cellular functions, including homeostasis, ion conductance, nutrient uptake and intercellular signalling (Drew et al, 2003;Midgett & Madden, 2007). For undertaking functional and structural studies, their overexpression is a necessary prerequisite, as the natural levels of most membrane proteins are low .…”

Section: Introductionmentioning

confidence: 99%

Improved membrane protein expression in Lactococcus lactis by fusion to Mistic

Kong

2013

Microbiology

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Difficulty overexpressing (eukaryotic) membrane proteins is generally considered as the major impediment in their structural and functional research. Lactococcus lactis possesses many properties ideal for membrane protein expression. In order to investigate membrane protein expression in L. lactis, we created a novel expression system by introducing Mistic, a short peptide previously identified in Bacillus subtilis, into L. lactis. The potential of this system was demonstrated in the overexpression of a eukaryotic membrane protein (pkjDes4) and a prokaryotic membrane protein (pkjLi), a newly isolated linoleate isomerase from Lactobacillus acidophilus. The expression levels reached up to 4.4 % and 45.2 % for pkjDes4 and pkjLi, respectively, which represented an exceptionally robust ability to overproduce membrane proteins. Moreover, the expressed pkjLi was functional, with its catalysing nature characterized for the first time in this species. Up to 0.852 mg ml "1 conjugated linoleic acid was obtained during the linoleic acid conversion catalysed by the recombinant lactococcal strains. In summary, we established a membrane protein expression system in L. lactis and examined its functionality. Our results demonstrate that the Mistic chaperoning strategy can be efficiently applied to L. lactis hosts and show its extraordinary capacity to facilitate the high-yield production of intractable membrane proteins.

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Breaking the bottleneck: Eukaryotic membrane protein expression for high-resolution structural studies

Cited by 83 publications

References 118 publications

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors

Rational and Irrational Approaches to Convince a Protein to Crystallize

Improved membrane protein expression in Lactococcus lactis by fusion to Mistic

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Breaking the bottleneck: Eukaryotic membrane protein expression for high-resolution structural studies

Cited by 83 publications

References 118 publications

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABAA and serotonin receptors

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABAA and serotonin receptors

Rational and Irrational Approaches to Convince a Protein to Crystallize

Improved membrane protein expression in Lactococcus lactis by fusion to Mistic

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High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors